Upsala Journal of Medical Sciences. 2009; 114: 193–205
REVIEW ARTICLE
Pulsatility of insulin release – a clinically important phenomenon
BO HELLMAN
Department of Medical Cell Biology, Uppsala University, Uppsala, Sweden
Abstract
The mechanisms and clinical importance of pulsatile insulin release are presented against the background of more than half a
century of companionship with the islets of Langerhans. The insulin-secreting b-cells are oscillators with intrinsic variations of
cytoplasmic ATP and Ca2+. Within the islets the b-cells are mutually entrained into a common rhythm by gap junctions and
diffusible factors (ATP). Synchronization of the different islets in the pancreas is supposed to be due to adjustment of the
oscillations to the same phase by neural output of acetylcholine and ATP. Studies of hormone secretion from the perfused
pancreas of rats and mice revealed that glucose induces pulses of glucagon anti-synchronous with pulses of insulin and
somatostatin. The anti-synchrony may result from a paracrine action of somatostatin on the glucagon-producing a-cells.
Purinoceptors have a key function for pulsatile release of islet hormones. It was possible to remove the glucagon and
somatostatin pulses with maintenance of those of insulin with an inhibitor of the P2Y1 receptors. Knock-out of the adenosine
A1 receptor prolonged the pulses of glucagon and somatostatin without affecting the duration of the insulin pulses. Studies of
isolated human islets indicate similar relations between pulses of insulin, glucagon, and somatostatin as found during perfusion
of the rodent pancreas. The observation of reversed cycles of insulin and glucagon adds to the understanding how the islets
regulate hepatic glucose production. Current protocols for pulsatile intravenous infusion therapy (PIVIT) should be modified
to mimic the anti-synchrony between insulin and glucagon normally seen in the portal blood.
Key words: ATP, calcium oscillations, diabetes, glucagon, insulin, islets of Langerhans, purinergic receptors, somatostatin
Introduction
For more than half a century pancreatic islets have
been intensely studied at the University of Uppsala.
After examining the mechanisms for alloxan destruction of the insulin-producing b-cells (1) and the
principles for dissemination of the endocrine pancreas
into islets (2), most of my attention has been paid to
the insulin secretory process. Early studies of isolated
islets made it possible to propose that glucose stimulation of insulin release is mediated by increase of the
cytoplasmic Ca2+ concentration ([Ca2+]i) in pancreatic b-cells (3). This idea was confirmed by direct
measurements of [Ca2+]i (4). Even more important,
use of ratiometric fura-2 technique demonstrated the
existence of oscillatory rises of [Ca2+]i (5,6) that
triggered 3–4 min pulses of insulin release (7).
A prerequisite for pulsatile release of islet hormones
is that the [Ca2+]i oscillations are entrained into a
common rhythm in the cells involved. Accumulating
data indicate that both individual cells and whole islets
behave as coupled oscillators (8,9). Like other limitcycle oscillators the b-cells are expected to synchronize
when the coupling signal is sufficient to overcome the
differences in natural frequencies. We imagine that
the synchronization emerges co-operatively, analogous
to phase transitions such as freezing of water or spontaneous magnetization of a ferromagnet (10,11). In
accordance with phase transitions, the alignment of
islet cell oscillations in time may be the counterpart of
the alignment of molecules in space.
This review presents the author’s views about the
mechanism and clinical importance of pulsatile insulin release with emphasis on recent contributions from
Correspondence: Professor Bo Hellman, Department of Medical Cell Biology, Biomedicum Box 571, SE-75123 Uppsala, Sweden. Fax: +46 184714059.
E-mail: [email protected]
(Received 17 September 2009; accepted 24 September 2009)
ISSN 0300-9734 print/ISSN 1502-4725 online 2009 Informa UK Ltd. (Informa Healthcare, Taylor & Francis AS)
DOI: 10.3109/03009730903366075
194
B. Hellman
our laboratory. Besides examining how b-cells generate pulses of insulin release it is discussed how these
cells co-ordinate their rhythmicity. The finding that
pulses of insulin are anti-synchronous to those of
glucagon adds to the understanding of the islet
regulation of hepatic glucose production.
Insulin pulses are triggered by intrinsic b-cell
rhythmicity of ATP and Ca2+
A number of studies have indicated periodic variations of circulating insulin in the peripheral blood
(12,13). Initially, these oscillations were supposed to
reflect pulsatile release of insulin generated by the
central nervous system. This idea was refuted after the
observation that insulin is released from the isolated
pancreas in a pulsatile fashion (14). The controversies
about the pacemaker for pulsatile insulin release were
settled after our observation that b-cells have the
intrinsic ability to generate 2–5-min [Ca2+]i oscillations resulting from periodic depolarization (5,6).
In 1969 we organized an international symposium
as a centennial of Paul Langerhans’ discovery of the
islets (15). At that time it was generally accepted that
glucose depolarizes the b-cells via its metabolism.
A few years later colleagues in Umeå provided the
first evidence that the glucose-induced depolarization
of the b-cells was mediated by suppression of the K+
permeability (16). The introduction of the patch
clamp technique (17) made it possible to demonstrate
that b-cells have K+ channels inhibited by cytoplasmic
ATP. The discovery of the KATP channel was made in
Oxford (18), but many of its properties were first
described in a thesis from Uppsala (19).
The key role of ATP for generation of insulin
release pulses is schematically illustrated in Figure 1.
The cytoplasmic concentration of ATP (20) as well as
the ATP/ADP ratio (21) are known to oscillate in
glucose-stimulated b-cells. The metabolism of glucose induces periodic rises of cytoplasmic ATP due to
oscillatory glycolysis mediated by the allosteric
enzyme phosphofructokinase-M (22). Increase of
ATP promotes insulin release by closure of the
Adenosine
CD73 (Ecto-5′-NT)
P1 receptor
Glucose
Glucose
AMP
Exocytosis
Closure of
KATP channels
Depolarization
Oscillatory
metabolism
generates
ATP
∆Ψ
Insulin
CD39 (E-NTPD)
ATP
ADP
Opening of
Ca2+channels
Ca2+
Ca2+
cAMP
P2 receptor
Figure 1. Model showing how glucose-induced oscillations of cytoplasmic ATP generate pulsatile release of insulin from a b-cell. Rhythmic
glycolysis triggers periodic rises of cytoplasmic ATP that inhibits a specific K+ channel. Resulting depolarization evokes oscillations of
cytoplasmic Ca2+ due to entry of the ion via voltage-dependent channels. The right part of the Figure shows that oscillatory rises of cytoplasmic
Ca2+, ATP, and cAMP evoke exocytosis of secretory granules containing insulin together with ATP, ADP, and AMP. After release from the bcell these nucleotides, degraded or not to adenosine by ectonucleotidases (CD39 and CD73), serve as regulators of pulsatile release of insulin
by binding to P1 and P2 receptors.
Pulsatility of insulin release
195
A.
Glucose 11 mM
400
0
Cytoplasmic Ca2+ (nM)
400
0
5 min
B.
Glucose 11 mM
400
a
a
0
400
b
b
0
400
c
c
0
5 min
Figure 2. Oscillations of cytoplasmic Ca2+ in two mouse b-cells lacking contact (A) and in three cells situated in an aggregate (B). Contacts
between the cells result in synchronization of the Ca2+ oscillations. The traces refer to the cells shown to the right.
KATP channels with subsequent depolarization and
influx of Ca2+ via voltage-dependent channels. Moreover, ATP sensitizes the secretory machinery to the
Ca2+ signal, an effect amplified by cyclic AMP derived
from ATP. Studies in our laboratory have shown that
glucose induces periodic variations of cyclic AMP and
plasma membrane phosphoinositide lipids (23–25).
Glucose generation of pronounced oscillations of
phosphatidylinositol 3,4,5-trisphosphate has been
attributed to periodic release of insulin stimulating
its receptors on the surface of the b-cells (26).
Besides acting as a cytoplasmic initiator of pulsatile
insulin release ATP is a component of the secretory
granules (Figure 1). After periodic release via exocytosis, ATP serves as an autocrine and paracrine messenger by activating purinergic P2 receptors (27). The
P2 receptors belong to two major families: the ionotropic ligand-gated ion channel P2X and the metabotropic G-protein-coupled P2Y. Studies in our
laboratory have shown that P2 receptors are important
regulators of pulsatile insulin release from the b-cells
(9,28,29). One of several mechanisms is periodic
activation of phospholipase A2 with generation of
arachidonic acid, a substance known to inhibit the
KATP channels (30). Extracellular ATP is degraded
by ectonucleotidases to adenosine, which binds to P1
196
B. Hellman
Cytoplasmic Ca2+ (nM)
A.
Methoxyverapamil 50 µM
600
300
0
5 min
B.
Methoxyverapamil 50 µM
a
Cytoplasmic Ca2+ (nM)
600
a
76 µm
b
0
600
b
98 µm
0
600
c
c
0
5 min
Figure 3. Transients of cytoplasmic Ca2+ in ob/ob mouse b-cells superfused with a medium containing 20 mM glucose and 20 nM glucagon.
A: Suppression of the Ca2+ entry with methoxyverapamil removes the Ca2+ oscillations, allowing the transients to start from the basal level.
B: Synchronized cytoplasmic Ca2+ transients in single cell/aggregates (shown to the right) exposed to methoxyverapamil.
receptors. These receptors have modulatory effects on
islet hormone release by affecting the amplitude and
duration of the pulses (29,31).
Individual islet cells generate spontaneous Ca2+
oscillations with different frequencies
It is difficult to study pulsatile release of insulin from
individual b-cells without disturbing the spontaneous
rhythm. The knowledge of the intrinsic rhythmicity
of b-cells is therefore based essentially on measurements of [Ca2+]i. The observation that glucose generates oscillations of [Ca2+]i in single b-cells was first
reported from Uppsala (32). The oscillatory frequency
differs considerably among b-cells lacking contact
(Figure 2A). However, when situated in aggregates
the b-cells are entrained into a common rhythm of
about 0.3/min (Figure 2B). The oscillatory activity is
critically dependent on sufficient oxygen supply during
the isolation of the b-cells and procedures to minimize
the exposure to UV light during the measurements of
[Ca2+]i. Periodic rises of [Ca2+]i were seen not only in
b-cells but also in glucagon-producing a-cells (33),
somatostatin-producing d-cells (33), and pancreatic
polypeptide-producing (PP) cells (34).
b-Cells communicate via diffusible factors
generating cytoplasmic Ca2+ transients
b-Cell oscillations of [Ca2+]i are sometimes superimposed with transients mediated by inositol 1, 4, 5
Pulsatility of insulin release
197
Glucose 20 mM
500
a
0
Cytoplasmic Ca2+ (nM)
500
d
b
b
0
500
a
c
c
100 µm
0
500
d
0
5 min
Figure 4. Co-ordination of [Ca2+]i oscillations in the four aggregates shown to the right. Most of the superimposed transients appear in
synchrony not only within but also among the aggregates. Modified from Grapengiesser et al. 2003 (8) with permission.
trisphosphate (IP3). Comparison of different animal
models revealed that b-cells from obese-hyperglycaemic mice (ob/ob) are unusual by generating numerous
[Ca2+]i transients (35,36). Studies of these animals
have been important for demonstrating that b-cells
can communicate via diffusible factors. After eliminating the background of slow oscillations (inhibition of
the Ca2+ entry), it was found that the transients propagate between adjacent b-cells lacking contact
(Figure 3). In the attempts to identify the messengers
involved, evidence was provided for regenerative
release of ATP (37) and NO (38).
We have tested whether the transients of [Ca2+]i
have a co-ordinating action on the oscillatory activity
in isolated b-cells (8). The experimental conditions
were designed to promote IP3 generation of transients
(ob/ob mouse b-cells exposed to 20 mM glucose and
20 nM glucagon). It was seen that b-cells/aggregates
superimposed with synchronized transients are
entrained into a common rhythm (Figure 4). The
superimposed transients had a co-ordinating action
on [Ca2+]i oscillations in b-cells separated by a
distance of < 100 mm, but not in those situated
> 200 mm apart. There are other pathways for
generating [Ca2+]i transients than those mediated
by IP3. Studies in our laboratory indicate prominent
b-cell transients of [Ca2+]i resulting from intermittent
entry of the ion (39,40). Entry of Ca2+ via rapidly
inactivating P2X receptors represents an attractive
alternative for generation of the [Ca2+]i rises supposed to entrain the glycolytic oscillator into a
common rhythm.
Synchronization of b-cells within and among
the islets in rodents
Parallel measurements of [Ca2+]i and release of insulin from single mouse islets support the idea that
glucose-induced oscillations of [Ca2+]i generate
pulses of insulin (7). The role of glucose is both to
induce [Ca2+]i oscillations (41,42) and to make the
exocytotic machinery more sensitive to the Ca2+ signal (43). Within the islets the b-cells are well coordinated, as indicated by the presence of synchronized [Ca2+]i oscillations and distinct pulses of insulin
release from the whole islet. There is a need for a
strong coupling force to overcome the differences in
198
B. Hellman
A.
Insulin
Glucagon
1000
150
100
500
0
Insulin (pM)
0
5 min
B.
Insulin
Glucagon (pM)
50
Glucagon
20
1500
1000
10
500
0
5 min
0
Figure 5. Relation between repetitive insulin and glucagon pulses during perfusion of rodent pancreas with 20 mM glucose. A: The pulses of
insulin are anti-synchronous to those of glucagon in rat pancreas. From Grapengiesser et al. 2006 (49) with permission. B: The pulses of
glucagon are prolonged compared with insulin in mice with knock-out of the adenosine A1 receptor. From Salehi et al. 2009 (31) with
permission.
the endogenous b-cell rhythm. A major part of this
coupling is mediated by gap junctions made of connexin-36 (44,45). Regenerative release of ATP and
London other messengers contribute to the co-ordination by propagating [Ca2+]i transients between
the b-cells. In very large islets the coupling mechanisms are insufficient to synchronize all b-cells, as
reported from measurements of [Ca2+]i in ob/ob
mouse islets (46).
Each islet is an oscillatory unit, generating 3–4-min
pulses of insulin release (47). Contrary to the coordination of b-cells within an islet, the synchronization of the islets in the pancreas requires a weak
coupling force due to similarities in pulse frequency.
The entrainment of the islets into the same oscillatory
phase is very efficient, as indicated by the distinct
pulses of hormone release from the perfused rat
pancreas (48–50). The co-ordination of the b-cells
from the different islets in the pancreas is supposed
to be mediated by neural input from local ganglia
(51,52). It was recently reported that repetitive
pulses of the neurotransmitter acetylcholine, contrary to ATP, have a synchronizing action on isolated mouse islets (53,54). Mathematical modelling
supported the idea that acetylcholine-induced
increase of [Ca2+]i resets the glycolytic oscillator.
However, there are reasons to believe that the
failure to demonstrate a synchronizing effect of
ATP was due to rapid desensitization of purinergic
P2 receptors.
Pulsatility of insulin release
199
Pulsatile release of islet hormones in rodents
β-cell
Mutual
synchronization
Somatostatin
α-cell
δ-cell
Figure 6. Model of cell interactions important for glucose generation of pulsatile hormone release from an islet. Dotted lines
indicate level of basal release before the rise of glucose. Glucose
generates simultaneous pulses of insulin and somatostatin release by
mutual synchronization of b- and d-cells. The oscillations of glucagon appear in anti-synchrony and have nadirs below the basal
level. Paracrine release of somatostatin from d-cells accounts for the
appearance of glucagon pulses 180 out of phase.
A.
Like b-cells, the glucagon-producing a-cells and the
somatostatin-producing d-cells have an intrinsic ability to generate oscillations of [Ca2+]i. Entrained into a
common rhythm these oscillations trigger pulses of
hormone release. Increase of glucose from 3 to 20
mM generated pronounced pulses of insulin, glucagon, and somatostatin from the perfused pancreas of
rats (48–50) and mice (31). The major component of
hormone release was pulsatile, irrespective of whether
the rhythmicity resulted in increase (insulin and
somatostatin) or decrease (glucagon) of average secretory rate. Remarkably, the pulses of glucagon were
anti-synchronous to those of insulin and somatostatin
(49,50). The presence of reversed cycles of insulin
and glucagon (Figure 5A) is much to the purpose,
since these hormones have counteractive effects on
the hepatic glucose production.
The mechanisms for regulation of pulsatile release
of islet hormones are far from elucidated. Enhanced
secretory rates of insulin (13,55) and glucagon (56)
are usually related to increase of the pulse amplitude.
Other studies have shown that glucose-induced oscillations of [Ca2+]i are transformed into sustained elevation, when single mouse b-cells or intact islets are
B.
Insulin release (pmol/g islet dry weight /sec)
30
20
10
0
60 sec
Figure 7. Insulin release from a human islet exposed to 11 mM glucose. A: Pulse observed with a sampling time of 17.5 seconds. B: The same
pulse analysed with a sampling time of 2.5 seconds.
200
B. Hellman
Glucose 20 mM
amol/min/islet
1200
Insulin
800
400
0
amol/min/islet
Glucagon
80
40
0
Somatostatin
amol/min/islet
8
4
0
0
20
10
30
Time (min)
Figure 8. Effects of raising glucose from 3 to 20 mM on the release of insulin, glucagon, and somatostatin from a batch of 15 human islets. The
hormones were measured in 30-second samples of the perifusate.
exposed to amino acids (57) or noxious agents (58).
A pertinent question is whether the anti-synchrony
between insulin and glucagon is removed by alterations of the pulse duration. The answer is yes
(Figure 5B). Knock-out of the adenosine A1 receptor
was found to prolong the pulses of glucagon and
somatostatin but not of insulin (31). Interestingly,
the pulsatile insulin release from rat pancreas persisted when the pulses of glucagon and somatostatin
were suppressed by a low concentration of an inhibitor of the purinergic P2Y1 receptor (50).
The observation that islet hormones are released as
pulses, with glucagon in anti-phase with insulin and
somatostatin, makes it necessary to reconsider previous ideas how a-, b-, and d-cells interact within
rodent islets. A tentative model is presented
in Figure 6. It is proposed that b- and d-cells are
entrained into a common rhythm due to mutual
synchronization mediated by gap junctions and
diffusible factors (ATP). The coupling between the
two types of cells is weak, as suggested by the preservation of the insulin oscillations after prolongation
(31) or removal (29,50) of the somatostatin pulses.
Our model proposes that somatostatin, a well established inhibitor of secretion, modulates the pulsatile
release of glucagon from the a-cells. The observation
that periods with a rise of somatostatin are related to
decrease of glucagon reinforces existing arguments
that d-cells have tonic inhibitory effects on a-cells
(59–61). Such a paracrine action may well explain
why glucagon pulses are in anti-phase with pulses of
somatostatin and consequently also with insulin.
Pulsatile release of islet hormones in man
Privileged with access to human islets for more than
15 years we now conclude that the mechanisms for
Pulsatility of insulin release
1200
80
800
40
400
0
Glucagon (amol/min/islet)
Insulin
Glucagon
Insulin (amol/min/islet)
201
0
Insulin
1200
Insulin (amol/min/islet)
8
800
4
400
0
Somatostatin (amol/min/islet)
Somatostatin
0
5
10
20
15
25
30
Time (min)
Figure 9. Relation between the repetitive release pulses of insulin and glucagon in the experiment shown in Figure 8. The insulin pulses are
anti-synchronous to the glucagon pulses (upper panel) and coincide with the somatostatin pulses (lower panel).
pulsatile release of islet hormones in man resemble
those in rats and mice. It was possible to show that
also human b-cells have specific K+ channels regulated by cytoplasmic ATP (28). The activity of these
channels varied with the same periodicity as the
depolarizing waves triggering the Ca2+ entry into
the b-cells (62). The similarity with rodents refers
not only to the [Ca2+]i rises that trigger insulin pulses
but also to kinetics of hormone release (28). Measurements of insulin release with high time resolution
revealed that secretory pulses from single islets can
be resolved into episodes of 10–20 seconds (Figure 7).
These episodes reflect the bursts of electrical activity
characteristic of b-cells situated in intact islets (63).
Species differences in the cytoarchitecture of the islets
make it important to analyse whether islet hormone
pulses in rodents have their counterparts in man. It
has been reported that [Ca2+]i oscillations are poorly
co-ordinated in b-cells (64) and totally asynchronous in
a-cells (65) within human islets. Moreover, uncertainties exist whether the insulin oscillations in man are
related to periodic variations of other islet hormones
(66–69). Our recent studies of isolated human islets
indicate that an increase of glucose from 3 to 20 mM
induces release pulses of insulin, glucagon, and
somatostatin at regular intervals (70). A representative
experiment is shown in Figure 8. The relation between the hormones was similar as found during perfusion of rodent pancreas (see above). Accordingly, the
pulses of insulin were anti-synchronous to those of
glucagon (Figure 9, upper panel) but coincided with
the somatostatin pulses (Figure 9, lower panel). Due to
the anti-synchrony there were > 20-fold variations of the
insulin/glucagon ratio during a pulse cycle (Figure 10).
202
Insulin/glucagon ratio (arbitrary units)
B. Hellman
30
20
10
10
20
30
60
70
80
Time (min)
Figure 10. Variations of the insulin/glucagon ratio during superfusion of 15 human islets with 20 mM glucose. Sampling was interrupted for
28.5 min in the middle of the experiment. The insulin/glucagon ratio is given in arbitrary units with the average of nadir values set to 1.0 (dotted line).
The glucose-induced generation of pulses resulted in
marked increase of time-averaged release of insulin and
somatostatin. In the case of glucagon the nadirs between
the pulses were lower than at 3 mM glucose, resulting in
a slight but significant suppression of average release.
Clinical aspects on insulin and glucagon
pulsatility
Pulsatile release of insulin into the portal vein generates 3–4-min oscillations, the amplitude of which is
markedly suppressed after passage through the liver.
The effects of insulin and other islet hormones are
critically dependent on their possibilities to rapidly
reach the target cells via fenestrations of the capillary
endothelium. Besides the pancreatic islet cells also the
liver cells are exposed to pronounced oscillations of
insulin and glucagon. It is open for discussion to what
extent target cells at other locations are directly
affected by the periodic variations of the insulin/
glucagon ratio.
A major advantage of periodic compared with continuous exposure to the hormones is to prevent downregulation of their receptors. Several studies indicate
that the cyclic variations of insulin and glucagon keep
the receptors on the liver cells up-regulated (71–75).
Pulse administration may be useful also in other
kinds of receptor-mediated diabetes therapy.
Attempts should be made to increase the number
of glucagon-like peptide-1 (GLP-1) receptors, which
are down-regulated in b-cells during hyperglycaemia
and overt diabetes (76). Indeed, supraphysiological
concentrations of GLP-1 have been found to correct
both the deficient release of insulin and the excessive
release of glucagon in type 2 diabetes (77). For
practical reasons, pulse stimulation of the GLP-1
receptor should be performed with a stable analogue
of GLP-1 (i.e. extendin-4).
Loss of regular oscillations of insulin is an early
indicator of diabetes (12,13). The starting-point for
treatment of diabetes is to investigate if the absence of
regular insulin periodicity can be attributed to deficient b-cell rhythm or co-ordination. Efforts should
be made to re-establish a normal periodicity. Interestingly, islets transplanted to the human liver have
been found to release insulin in pulses (78). It is
possible to generate regular insulin oscillations in
the portal vein by bolus injections of the hormone
into a hand or forearm vein. Sessions of pulsatile
intravenous infusion therapy (PIVIT) have been
reported to counteract renal and neural complications
in diabetes (79–81). Recent progress in the understanding of islet hormone release urges for modifications of the PIVIT protocol to mimic the reversed
pulses of insulin and glucagon.
Acknowledgements
I would like to express my gratitude to my colleagues
at the Department of Medical Cell Biology, Biomedical Center, for stimulating collaboration. Progress in
the understanding of hormone secretion from human
islets became possible after Olle Korsgren’s
Pulsatility of insulin release
establishment of The Islet Transplantation Center at
the Rudbeck Laboratory. Special thanks for many
years of companionship in the world of the islets to:
Erik Gylfe, the present Head of the Department, who
has been of vital importance for the research both in
Umeå and after my return in 1976 to Uppsala, Eva
Grapengiesser who was the first to demonstrate the
intrinsic Ca2+ rhythmicity of the b-cells, Anders
Tengholm for keeping me updated in molecular
cell biology, and Heléne Dansk, whose enthusiasm
and skilfulness made it possible to reveal many secrets
of the b-cells.
The author is the recipient of the Rudbeck Award
2008.
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